Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: A single-step approach
Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: A single-step approach
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DOI：
10.1373/clinchem.2004.031153
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发表时间：
2004-01-01
影响因子：
9.3
通讯作者：
Wong, LJC
中科院分区：
文献类型：
--
作者：
Bai, RK;Wong, LJC
背景资料：A3243 G线粒体tRNA Leu（UUR）点突变导致线粒体肌病、脑病、乳酸酸中毒和卒中样发作（MELAS）综合征，这是最常见的线粒体DNA（mtDNA）疾病，也见于母系遗传性糖尿病和耳聋综合征（MIDD）患者。为了将疾病表现与突变负荷相关联，有必要测量A3243 G mtDNA突变的百分比。方法：为了可靠地定量低比例的突变mtDNA，我们开发了实时扩增难治突变系统定量PCR（ARMS-qPCR）测定。我们用含有已知比例的突变型A3243 G mtDNA的实验样品验证了该方法，该突变型A3243 G mtDNA是通过混合已知量的含有野生型或突变型序列的克隆质粒DNA产生的。发现实验样品中突变体A3243 G的比例的预期值和观察值之间的相关系数为0.9995。证明了PCR-限制性片段长度多态性（RFLP）分析和实时ARMS-qPCR之间的一致结果。然而，后一种方法对于检测低百分比的突变异质性要敏感得多。结论：实时ARMS-qPCR方法可快速、可靠、一步定量检测异质性突变mtDNA。(C)2004年美国临床化学协会。
Background: The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation.Methods: To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences.Results: A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR-restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations.Conclusions: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA. (C) 2004 American Association for Clinical Chemistry.